IE53210B1 - Derivatives of m.139,603 useful as growth promoters - Google Patents

Abstract

Novel ether, ester, urethane, thiourethane and carbonate derivatives of the ruminant growth promoters, M. 139,603, M. 139,603-aldehyde and oxime etc. derivatives thereof, all of which are effective in reducing the proportion of methane produced by ruminal fermentation, and increasing the proportion of propionic acid at the expense of acetic acid in the rumen contents; together with processes for their manufacture, compositions containing them, and their use in increasing growth and/ or feed efficiency in ruminant animals.

Description

This invention relates to derivatives of the animal growth promoter known as M.139,603. These derivatives are effective in reducing the proportion of methane produced by ruminal fermentation, and increasing the proportion of propionic acid in rumen fluid, and are therefore believed to possess growth promoting properties in ruminants, it being well known that other chemical compounds which reduce methane and increase propionic acid levels in the rumen result in increased growth rates when fed to ruminants such as cattle and sheep.

The compound known as M. 139,603, which is a metabolite obtained from the aerobic culture of Streptonyces longisporoflavus, is described and claimed in GB-A-2,021,013, and has the structure I, R1 = methyl, R^ = hydroxymethyl.

The invention provides a derivative of M.139,603 of the formula I wherein:M+ is an alkali metal, alkaline earth metal, ammonium, alkylammonium or hydroxyalkylammonium cation; R1 is a hydrogen atom, a 1-6C alkyl radical or a phenyl (1-6C)alkyl radical optionally substituted by halogen, cyano or 1-8C alkyl, alkoxy, halogenoalkyl, halogenoalkoxy or alkanoylamino radicals; and 3 3 R is a radical of the formula -CH2OR wherein R is a 1-6C alkyl, 3-6C alkenyl, 3-6C alkynyl, 2-6C alkoxycarbonyl or 1-6C alkylamino-(2-6C)alkyl radical or a phenyl(1-6C)alkyl radical bearing an optional substituent as defined above; or a radical of the formula -CH^O.COR4 wherein R4 is an alkyl, cycloalkyl, (cycloalkyl)alkyl, halogenoalkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, mono- or di-alkylaminoalkyl, alkoxycarbonylalkyl, carboxyalkyl, carbamoylalkyl, N- alkylcarbamoylalkyl or Ν,Ν-dialkylcarbamoylalkyl radical wherein each alkyl contains up to 6 carbon atoms, and wherein, in each complex radical containing an alkyl part, the said alkyl part contains 1 to 6 carbon atoms, and wherein each cycloalkyl radical or eyeloalkyl part of a (cycloalkyl,alkyl radical contains 3 to 7 carbon atoms, or a phenyl, naphthyl or phenyl(1-6C)alkyl radical each bearing optional substituents as defined above; or a radical of the formula -CH2O.CO.COR5 wherein R5 is an amino, mono- or di-(1-6C)alkylamino, or 1-6C alkoxy radical, or a phenyl, naphthyl or phenyl(1-6C)alkyl radical each bearing optional substituents as defined above; or a radical of the formula -CH_0.CX.NR6R7 wherein X is an oxygen or sulphur atom and R and R , which may be the same or different, are each a hydrogen atom, a 1-6C alkyl radical or a phenyl, naphthyl or phenyl(1-6C)alkyl radical each bearing optional substituents as defined above; or when R is a hydrogen atom, R is a hydroxymethyl radical.

A particular value for any of R1, R2, R3, R4, R5, fi 7 R or R when it is an alkyl radical, or when it is a complex radical containing an alkyl part, is, for example, such an alkyl radical which contains 1 to 3 carbon atoms, or such a complex radical wherein the alkyl part contains 1 to 3 carbon atoms.

A particular value for R when it is an alkenyl or alkynyl radical is, for example, such a radical of 3 carbon atoms.

Particular cations M+ are, for example, the sodium, lithium, potassium, calcium, magnesium, zinc, ammonium, mono-, di-, tri- and tetra-alkylammonium ions wherein each alkyl is of 1 to 10 carbon atoms, and mono-, di- and tri- (hydroxyalkyl)ammonium ions wherein each hydroxyalkyl is of 1 to 10 carbon atoms.

Particular compounds of the invention which are preferred for increasing the proportion of propionic acid in rumen fluid are those compounds of the formula I wherein:1 2 R is hydrogen and R is hydroxymethyl; is methyl is methyl is methyl is methyl is methyl is methyl is methyl and R is and R2 is and R2 is and R2 is and R2 is and R2 is and R2 is methoxymethyl; propargyloxymethyl; benzoyloxymethyl; 2-bromobenzoyloxymethyl; chloroacetoxymethyl; ethoxalyloxymethyl; and N-methylthiocarbamoyloxymethyl.

The compounds of the invention may be prepared by processes which are known in themselves for the manufacture of chemically analogous compounds. Thus, the following processes are provided as a further feature of this invention:2 (a) for those compounds wherein R is a radical of the formula -CH2OR as defined above, the reaction of a compound of the formula I wherein has any of the meanings defined 2 above and R is a hydroxymethyl radical., with a compound of the formula R3Z, wherein R3 has the meaning defined above and Z is a leaving group, for example a halogen atom, such as chlorine or bromine, or a mesyl or tosyl group, in the presence of a strong base, for example sodium hydride or a sodium lower-alkoxide such as sodium methoxide or sodium ethoxide; for those compounds wherein R1 is other than 4 hydrogen, and R is a radical of the formula -CE^O.COR , -CH2O.CO.COR5 or CH2O.CX.NR6R7 as defined above, (b) 532 10 the reaction of a compound of the formula I wherein R1 is an alkyl or optionally substituted phenylalkyl radical and R is a hydroxymethyl radical, with an acylating agent derived from a carboxylic acid of the formula R4.C00H or R5 CO.COOH wherein R4 and R5 have the meanings defined above, for example an acyl halide, such as the chloride, bromide or iodide, with an acid anhydride, with a mixed anhydride, or with a carboxylic 4 5 acid of the formula R .COOH or R CO.COOH in the presence of a carbodi-imide, for example dioyolohexylcarbodiio -imide; whereinafter:- for those compounds wherein R is a radical of the formula -CH2O.COR4 in which R4 is a hydroxyalkyl radical, a compound of the formula I wherein R2 is a radical of the formula -CH20.C0R4 in which R4 is a (t-alkoxy)-alkyl radical obtained as described above, is reacted with an acid, for example ethanolic hydrogen chloride; or for those compounds wherein R is a radical of the formula -CH2O.COR4 in which R4 is an alkylaminoalkyl or dialkylaminoalkyl radical, the corresponding aminoalkyl compound obtained as described above, is reacted with an alkyl halide, for example an alkyl bromide or alkyl iodide; or for those compounds Ο A wherein R is a radical of the formula -CHjO.COR in which R4 is an alkoxycarbonylalkyl radical, the corresponding carboxyalkyl compound obtained as described above, is reacted with a diazoalkane, or a salt of the corresponding carboxyalkyl compound,obtained as described above, is reacted with an alkyl halide, for example an alkyl bromide or alkyl iodide; or for those compounds wherein R is a radical of the formula -CHjO.COR in which R is a carbamoylalkyl, N-alkylcarbamoylalkyl or N,N-dialkylcarbamoylalkyl radical, a corresponding carboxyalkyl or alkoxycarbonylalkyl compound obtained as described above, Is reacted with ammonia or a mono- or di-alkylamine respectively; or for those compounds wherein R2 is a radical of the formula -CH2O.CO.COR5 in which R5 is an amino, monoalkylamino or dialkylamino radical, a c corresponding compound wherein R is a hydroxy or alkoxy radical, obtained as described above, is reacted with ammonia or a mono- or di-alkylamine respectively; or for those compounds wherein R is a radical of the formula 7 6 7 -CHjO.CX.NR R wherein R is a hydrogen atom and X and R have the meanings defined above, a corresponding compound of the formula I in which R2 is a hydroxymethyl radical obtained as described above, is reacted with an •7 isocyanate or isothiocyanate of the formula R .NCX; or for those compounds wherein R is a radical of the formula -CH,O.CX.NR6R7 wherein X has the meaning defined 6 7 above and either one or both of R and R is other than a hydrogen atom, the reaction of a corresponding compound 6 7 wherein either or both of R and R is a hydrogen atom, obtained as described above, is reacted with a halogen 6 7 compound R .halide or R .halide; or for the compound wherein R is a hydrogen atom and R is a hydroxymethyl radical, the culturing of Streptomyces lonoisporoflavus NCIB 11426 or a variant or mutant thereof in an aqueous nutrient medium, containing a source of assimilable carbon, under shaken aerobic conditions at a temperature of between 22 and 32°C., extracting the fermentation mixture with a water-immiscible organic solvent, and isolating from the organic extract the product which has Rp = 0.43 on thin-layer chromatography on silica plates (Merck's Kieselgel 60F-254 - trade mark), 0.25mm. thickness eluting with a mixture of diethyl ether, methanol and formic acid in the proportion 95:4:1 by volume.

The micro-organism S.longisporoflavus NCIB 11426, which, is used in the process above is a known micro-organism which is available to the public without any restriction, upon payment of any required fee, from the National Collection of Industrial Bacteria, Ministry of Agriculture, Fisheries and Food, Torry Research . Station, 135 Abbey Road, Aberdeen AB9 8DG, Scotland.

As stated above, the compounds have the effect of increasing the proportion of propionic acid in rumen fluid, and in particular increase the proportion of propionic acid at the expense of methane and/or acetic acid. This is known to be a desirable effect in ruminant nutrition, because propionic acid is a much more efficient precursor of glucose, from which the animal derives its energy and growth, than is acetic acid; while that part of the animals' food intake vzhich is converted to methane is simply lost to the animal, the methane being excreted by eructation. Thus, the modification of rumen metabolism achieved by the compounds of the invention is a most useful effect, and is believed to increase the rate of growth and the food conversion efficiency of ruminant animals. & Thus, according to a further feature of the invention there is provided a method for use in the husbandry of domestic ruminant animals, for increasing the efficiency of their food conversion, which comprises orally administering to the animals a compound of the invention as described above.

In the method of the invention, the compounds of the invention are preferably orally administered to the animals as a supplement to their normal diet, that is to say, in admixture with salt-licks, dissolved in the drinking water or, for young animals such as lambs or calves, dissolved in whole milk or skim milk. The compounds of the invention are incorporated into food, feedblocks, salt-licks, drinking water, whole milk and skim milk to such an extent that each treated animal will ingest from 0,01mg./kg. body weight to 30mg./kg. body weight per day, preferably from O.Olmg./kg. to lOmg./kg. per day, of a compound of the invention.

The compounds of the invention may alternatively be orally administered to animals in the form of a slowrelease, intra-ruminal pellet or bolus, such that the animal will absorb a similar quantity per day of a compound of the invention.

The animals may receive the compound of the invention for substantially the whole of their growing period, or for only a part of their growing period, for example the early part and/or the period leading up to slaughter. The increase in growth rate achieved by practising the method of the invention enables animals reared for meat to be brought to market weight or slaughter weight in a shorter growing period than normal or it enables heavier animals to be produced at the end of the normal growing period. The improved efficiency of food conversion achieved by practising the method of the invention enables treated animals to reach any desired weight while consuming less food than untreated animals grown to the same weight. At optimum growth promoting inclusion levels, no indication of any toxic effect due to the compounds of the invention is observed.

According to a further feature of the invention, there is provided a composition which comprises a compound of the invention, together with a solid or liquid, edible non-toxic diluent or carrier, A suitable liquid diluent or carrier is, for example, drinking water, whole milk or skim milk.

A suitable solid, edible, non-toxic diluent or carrier may be, for example, a conventional nutritionally balanced ruminant feedstuff, for example a typical cattle or sheep diet consisting of cereal products, such as barley meal, maize meal or wheat feed, nut and seed products, such as decorticated ground nut cake or cotton seed cake, or extracted cotton seed cake, together with minor amounts of, for example feather meal, seaweed meal, bone meal, bone flour, chalk, salt, urea, molasses, vitamines and trace minerals? or it may be an inert solid diluent or carrier of no energy value, for example kaolin, talc, calcium carbonate, fuller's earth, attapulgus clay, ground oyster shells or ground limestone; or it may be starch or lactose. li The composition of the invention may take the form of a supplemented feedstuff for direct feeding to animals, in which case it will contain from 5 ppm to 3000 ppm of the compound of the invention in admixture with a conventional ruminant feedstuff; or it may take the form of a concentrated premix for dilution with a conventional ruminant feedstuff to produce a supplemented feedstuff suitable for direct feeding, and such a premix will contain from 0.3% w/w to 50% w/w of the compound of the invention in admixture with either a conventional, nutritionally balanced ruminant feedstuff, an inert solid diluent of no energy value, for example ground limestone, or starch or lactose.

According to a further feature of the invention there is provided a process for the manufacture of a solid composition of the invention which comprises uniformly mixing a compound of the invention with a solid, edible, non-toxic diluent or carrier.

The compound of the formula I is preferably serially diluted with the diluent or carrier in two or more successive stages, to ensure even mixing.

The invention is illustrated by the following Examples:25 Example 1 M.139603 (l.Og.) was dissolved in dichloromethane (40ml.), acetyl chloride (5ml.) was then added and the mixture allowed to stand in a stoppered flask at room temperature over the weekend. The solution was then filtered and the filtrate was evaporated to dryness. The resulting gum was examined on thin layer chromatography (TLC), which showed that approximately 90% of a new product had formed. The gum was dissolved in a little acetone and spread onto two preparative TLC plates (Merck ART 5717 - 40cm. x 20cm. x 2mm) which were developed in chlorofornyTnethanol/formic acid (90:5:5).

Two bands Rp = 0.46 and 0.54 were removed from the plate and eluted with ethyl aeetate/methanol (95:5) to give respectively 490mg. of a new product, and 320mg. of a mixture of the new product and the starting material, M.139,603. The mixed material of Rp = 0.46 was repurified in a similar manner to give 192mg. of single spot product; this material was combined with the previously eluted product to give a total of 682mg. of M.139,603 acetate, having the NMR spectrum, in deuterochloroform as shown in Figure 1.

In a similar manner, using 2-bromobenzoyl chloride in place of acetyl chloride, there was obtained:M.139,603 2-bromobenzoate (I, R1 = CHg, R2 = CH2O.CO(2-Br 2o CgH^) . Analysis: Calculated for C42H56°9BrNa·2H2O: C, 59.8; H, 7.1%. Found C,59.7; H,6.8%. Accurate mass measurement of mass spectrum molecular ion: calculated for C42H5gOg79BrNa: 806.2988; Found: 806.3006.

Example 2 To a stirred solution of the sodium salt of M.139,603 (O.624g., l.Ommote) in methylene chloride (2.0ml.) was added ethoxalyl chloride (0.5ml. 4.5mmole), and the mixture was stirred at room temperature for 64 hours. The solvent was evaporated under reduced pressure and the residue was treated with 50% aqueous ethanol containing sodium hydrogen carbonate to destroy the excess acid chloride. This mixture was diluted with water and extracted twice with ether, and the combined ether extracts were washed with water, dried (anhydrous magnesium sulphate) and concentrated to dryness under reduced pressure. The residue was chromatographed on silica gel (Merck 7734) using toluene-acetone (4:1) as eluant. Evaporation of the appropriate fractions gave the ethoxalyl ester of M.139,603 (I, R1 = CH-j, R2 = CHjO.CO.COOC^Hg) as a white froth. Calculated for C^gH^O-j^Na : C, 64.6, H, 7.9%. Found C,64.8; H, 8.1%. Accurate mass measurment of mass spectrummdecular ion: Calculated for C3gH57O11Na: 724.3799; found : 724.3796.

In a similar manner, the following ester derivatives of M.139,603 were prepared from the sodium salt of M.139,603 and the appropriate acid halide, Structures were confirmed by accurate mass measurement of the mass spectrum molecular ion. ί. 4 R2 Accurate Mass Measurement of molecular ion Analysis -CHjO.CO.CHjBr1 Calculated for C37H54 Br09Na 744.2850 Found: 744.2791 Calculated for C37H54BrO9Na! C, 59.6? H, 7.2; Br, 10.7%. Found: C, 59.9; H, 7.5; Br, 9.9%. -ch2o.co.ch2ci2 Calculated for C37H5435clO9Na 700.3354 Calculated for C37H54C109Na: C, 63.4; H,7.7; Found: 700:3383 Cl, 5.1%. Found: C, 63.0; H, 7.8? Cl, 5.4% -CH20.C0.CgH52 Calculated for Calculated forC42H57°9Na 728.3901C42H57°9Nai*H2° C,68.4? H,7.9%. Found: 728.3938 Found: C,68.5? H, 7.8%.

Footnotes 1. Used 6.0 equivalents of acid chloride. 2. Used 3.8 equivalents of acid chloride, and stirred at room temperature for 20 hours.

Example 3 To a stirred solution of the sodium salt of M.139,603 (l.Og., 1.60 mmole) in toluene (,10ml.) was added succinic anhydride (0.18g. 1.8 mmole) and triethylamine (0.26ml,, 1.9 mmole). The mixture was heated at reflux temperature for 20 hours, then cooled, and the solvent was evaporated under reduced pressure, The residual gum was dissolved in ether (100ml.) and the solution was extracted with saturated sodium carbonate solution (2 x 100ml.). The combined aqueous extracts were acidified with 2N hydrochloric acid and extracted with ether (2 x 70ml.), and the combined ether extracts were dried (anhydrous magnesium sulphate) and concentrated under reduced pressure. The residual gum was purified by chromatography on silica gel (Merck 7734) using ethyl acetate as eluant. The appropriate fractions were combined and concentra ted under reduced pressure to give the monosuccinyl ester of M.139,603 (X, R1 = CH3, R2 = CH2O.CO.CH2CH2COOH) as a white froth. Analysis: Calculated for C3gHg70^^Na2H20 : C, 61.6; H, 8.0%. Found: C, 61.3; H, 7.8%. Accurate mass measurement of mass spectrum molecular ion: calculated for C^gHg^O^^Na 724.3799; found 724.3810.

Example 4 To a stirred solution of the sodium salt of M.139,603 (312mg., 0.5 mmole) in toluene (2ml.) was added methyl isocyanate (0.18ml., 3.0 mmole) and, after heating the solution at 100° for 2 hours, the solvent was evaporated under reduced pressure. The residue was purified by chromatography on silica gel (Merck 7734) using ethyl acetate as eluant. The appropriate fractions were combined, and concentrated under reduced pressure to give the N-methylcarbamoyl ester of M.139,603 (I, R1 = N-methylcarbamoyl ester of M.139,603 (I, R1 = CH,, J R = CHjO.CONH.CHg) as a white froth. Analysis: Calculated for C-^HggNOgNal^O; C, 62.7; H, 8.3; N, 2.0%. Found: C, 62.7; H, 8.3; N, 1,8%, Accurate mass measurement of mass spectrum molecular ion: calculated for C37H56NOgNa 681.3853; found 681.3825.

In the similar manner, the following urethane and thiourethane derivatives of M.139,603 were prepared from the sodium salt of M.139,603 and the appropriate isocyanate or isothiocyanate. Structures were confirmed by accurate mass measurement of the mass spectrum molecular ion.

R2 Accurate Mass Measurement of molecular ion Analysis -C^O.CS.NH.C^1'2 Calculated for C37H56N08SNa: C, 62.8; H, 7.9; N, 2.0; S, 4.5%. Found: C, 62.8; H, 8.0; N, 1.8; S, 4.1% -CH20. CO. NH toy 3CH32 Calculated for Calculated forC4OH62NO9Nai 723.4310C4OH62NO9Na: C' 66.4; H, 8.6; N, Found: 1.9%. 723.4323 Found: C, 66.0; -CHjO. CO.NH-^^-Br3'4 H, 8.8; N, 2.3%. Calculated for C42H57N09BrNa^H20 : C, 60.6; H, 7.0; N, 1.7; Be, 9.6%. Found: C, 60.3; H, 6.8; N, 1.6; Br, 9.7% Footnotes 1. Used 7 equivalents of isocyanate 2. Heated at 100° for 18 hours 3. Used 1.2 equivalents of cyanate 4. Stirred at room temperature for 4 days.

Example 5 Μ. 139,603 (95mg.) was dissolved in ethyl acetate (10ml.) and phenyl isocyanate (30μ1.) added. The course of the reaction was followed by TLC and was shown to be essentially complete after standing overnight.

The solution was then spread onto a preparative TLC plate (Merck ART 5717, 20cm. x 20cm. x 2mm.) and developed in chloroform/methanol/formic acid (90:5:5).

Two bands, Rp = 0.48 and 0.58 were removed and eluted from the silica with ethyl acetate/methanol (95:5). Evaporation to dryness of the band, Rp = 0.58 containing the product, gave a colourless gum weighing 87mg. which was then crystallised from ether to give the N-phenylcarbamoyl ester of M.139,603 (I, R2 = CH^, R2 = CH2O.CO.NHPh), initially melting at 160-165°C., then resolidifying, and remelting at 2O3-2O5°C.

Example 6 To a stirred suspension of sodium hydride (0.05g., 2.1 mmole) in dry dimethylformamide (20ml.) and under an atmosphere of argon was added the sodium salt of M.139,603 (O.624g., 1.0 mmole). After stirring for hour at room temperature, methyl iodide (0.14ml., 2.2 mmole) was added and stirring was continued for a further hours. The reaction mixture was poured into water (100ml.) and the aqueous solution extracted with ether (2 x 50ml.). The combined ether extracts were washed with water (2 x 50ml.), dried (anhydrous magnesium sulphate) and concentrated under reduced pressure to give an oil which solidified on standing. Further drying under reduced l 2 pressure gave M.139.603 methyl ether (I, R = CH3, R = CH2OCH3) as a white froth. Analysis: Calculated for C36H55°8NalH2O: C' 65·9'· H' 8’7%, Found: C, 66.1; H, 9.0% Accurate mass measurement of mass spectrum molecular ion: calculated for C^Hg^OgNa 638.3795; found 638.3795.

In a similar manner, the following ether and carbonate derivatives of M. 139,603 were prepared from the sodium salt of M.139,603 and the appropriate alkyl, alkynyl or aryl bromide. Extractions were performed using ethyl acetate instead of ether and the products were further purified by chromatography on silica gel (Merck 7734) using ethyl acetate as the eluant. Structures were confirmed by accurate mass measurement of the mass spectrum molecular ion.

R2 Accurate Mass Measurement of molecular ion Analysis -ch2och2.c6h5 Calculated for C42H59°8Na : 714.4108 Found: 714.4118 Calculated for C42H59°8Na H2° C, 68.0; H, 8.4%. Found: C,68.O; H, 8.4%. Found: C, 68.1; H, 8.6% -CH2OCH2.C=CH Calculated for C38H55°8Na: 662.3795 Found: 662.3788 Calculated for C38H55°8Na °·5 H2° C, 68.0; H, 813%. Found: C, 68.3; H, 8.5% -CH2O(CH2)3CH3 Br Calculated for C39H61°8Na! 680.4265 Found: 680.4260 Calculated for C39H61°8Na °·5 H2° C, 67.9, H, 9.0% Found: C, 68.3; H, 9.1% -ΟΗ-ΟΟΗ,-γλ Calculated for Calculated forC42H5879BrO8Na 5 792.3203 Found : 792.3214C42H58BrO8Na 2-5H2° C, 60.1; H, 7.5; Br, 9.5%. Found: C, 60.2; H, 7.0; Br. 9.8% R2 Accurate Mass Measurement of molecular ion Analysis. -ΟΗ,Ο.ΟΟ.ΟΟ,Ης Calculated for Calculated forC38H57°10Na;C38H57°lONa 1H2° 696.3898 C, 63.9; H, 8.3%. Found: 696.3850 Found: C, 63.8; H, 8.2% -CHjOiCI^IjNiCH;,),1 Calculated for Calculated forC39H62NO8Na:C39H62NO8Na 2-5¾0 695.4342 C, 63.2; H, 9.0; Found: 695.4373 N, 1.9%. Found: C, 63.4; H, 8.5; N, 1.7% Footnote Eluted from silica with methylene chloride/ methanol (9:1).

Example 7 To a stirred solution of the sodium salt of M.139,603 (0.40g., 0.64 mmole) in petroleum ether (b.p. 60-80°) (40ml.) was added activated manganese dioxide (3.2g.). After stirring for 20 hours at room temperature, the reaction mixture was filtered and the filtrate was concentrated to dryness under reduced pressure. The residue was chromatographed on silica gel (Merck 7734) using ethyl acetate as the eluant and the appropriate fractions were concentrated under reduced pressure to give M.139,603 aldehyde (I, = CH3, R2 = CHO) as a white froth. Analysis: Calculated for C35H5]O8NaH2O. C, 65.6; H, 8.3%. Found: C, 65.9; H, 8.5%. Accurate mass measurement of mass spectrum molecular ion: calculated for C35H51OgNa 622.3482; ::0 found 622.3431.

Example 8 To a stirred solution of the sodium salt of M.139,603 aldehyde (0.10g., 0.16 mmole) in ethanol (5ml.) was added sodium acetate (0.log., 1.22mmole) and hydroxylamine hydrochloride (0.10g., 1.44ramole). The mixture was heated at reflux for % hour, cooled and filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was chromatographed on silica gel (Merck 7734 - trade mark) using acetone/60-80° petroleum ether (1:1) as eluant, and the appropriate fractions were concentrated under reduced pressure to give M.139,603 aldehyde oxime (I, R1 = CHg, R2 = CH=N0H) as a froth. Analysis: Calculated for C35H52°8NNa^2° C, 65.0; H, 8.2; N, 2.2%. Found C, 65.1; H, 8.6; N, 2.3%. Accurate mass measurement of mass spectrum molecular ion: calculated for CggHggOgNNa 637.3561; found 637.3591.

Example 9 Mother liquors from the crystallisation of M.139,603, obtained from the fermentation of Streptomyces longisporoflavus NCIB 11426 as described in United Kingdom Patent Specification No. 2027013A, were chromatographed on preparative thin layer chromatography (P.T.L.C.) plates (Merck ART 5717, 40cm. x 20cm. x 2mm.) using ether/glacial acetic acid (98.2) as the eluant.

The band at Rp = 0.40, corresponding to M.139,603, was discarded, but a second band at Rp = 0.23 was removed from the plates and eluted with ethyl aeetate/methanol (95:5) to give a viscous brown oil. This material was further purified by repeating the P.T.L.C. using the solvent system chloroform/methanol/formic acid (90:5:5). The band, Rp = 0.33, was removed from the plates, and the product, demethyl - M.139,603 (I, R^ = H, R2 = CHgOH), .‘21 was crystallised from petroleum ether (b.p. 6O-8O°C.), m.p. 156-158°C.

Example 10 Premixes suitable for dilution with an animal feedstuff may be manufactured by incorporating 10, 25, 50, 100 or 250mg. of the ethoxalyl ester of M. 139,603 in ground limestone so that the final weight of the premix is 500g.

Similar premixes may be manufactured using equivalent quantities of any other compound of the invention.

Example 11 An animal feedstuff suitable for direct feeding to ruminants may be manufactured by intimately mixing 500g. of a premix, obtained as described in Example 10, with 999.5mg. of a typical cattle feedstuff, to obtain a ruminant feedstuff containing 10, 25, 50, 100 or 250g. of the ethoxalyl ester of M.139,603, or any other compound of the invention, per metric ton, according to the concentration of the active ingredient in the premix used.

Suitable cattle feedingstuffs are :- Dairy Cake cwt kg Barley meal 512.5 Mai2e meal 1 50 Decorticated ground nut cake Ί J. 50 Decorticated cotton seed cake 1 50 Extracted cotton seed cake 1 50 Wheat feed 3 150 Feather meal 1 4 12.5 Seaweed meal 1 12.5 Bone meal 7-. 1 12.5 Chalk 7 1 12.5 1-» ο Dairy Cake (continued) Molasses Vitamins and trace mineral mix cwt 1¾ 1 4 k^ 75 12.5 20 1000.0 Beef Cube cwt kg^ Barley meal 11 550 Wheat feed 4 262.5 Decorticated ground nut cake I 4 12.5 Extracted ground nut cake 421bs. 18.75 Bone flour 1 4 12.5 Chalk 421bs. 18.75 Salt 141bs. 6.25 Molasses 2 12.5 Urea 1 4 12.5 Vitamins and trace mineral mix 141bs. 6.25 20cwtJ.000.00 Exainple 12 The ability of a compound of the invention to inhibit the production of methane in the rumen of ruminant animals, and to increase the proportion of propionate to acetate and butyrate (P/A + B) in the volatile fatty acids (VFA) produced, without at the same time depressing the overall digestive process, is demonstrated as follows:Rumen fluid is collected on a regular routine 25 basis from two steers, which are fed on the same hay-plus53210 Ϊ3 concentrate diet. Sampling time is standarised as far as possible, and the fluid from the two animals is pooled on a 50/50 basis. Large particulate matter is removed by filtering the pooled fluid through four layers of muslin cloth. The filtrate is then diluted in the ratio of one volume of filtrate to three volumes of an artificial rumen fluid (prepared as described by G.L.

Bales et al., Journal of Dairy Science, 1976, volume 59, page 1850, but omitting acetic acid), and the pH of the mixture is adjusted to 6.9-7.0 with saturated aqueous sodium carbonate solution. Aliquots (50ml.) of this mixture are dispensed into 100ml. conical flasks containing dried ground hay (0.5g.), and each flask is used to test a test compound at a particular concentration.

The test compound is added to the conical flask as a solution in ethanol, the flask is flushed with carbon dioxide gas, stoppered with a suba-seal, and incubated at 39°C. for 15-16 hours. After one hour, a narrow bore needle is inserted through the suba-seal to relieve the gas pressure, and the needle is withdrawn 30 minutes before the incubation is ended. Fermentation is then stopped by placing the flask in ice, and after 15 minutes cooling, the gas over the liquid is analysed for methane by gas chromatography. The flask contents are then filtered through a previously dried and weighed sintered glass funnel, the funnel is oven-dried and, by difference, the weight of hay digested is determined.

(Blank flasks containing no hay are treated similarly to determine non-cellulose residue). Three samples of the filtrate are analysed by gas chromatography for VFAs, and by comparison with the previously determined, preincubation level of VFAs, the net VFAs (acetate, propionate and butyrate) produced during the incubation is determined.

The following results were obtained (a negative control in which no test compound is employed is also included):- R1 R2 3ug./ml. 30jig./ Methane % of control P/A+B % of control Methane % of control P/A+B % of control ch3 CH2O.COCH3 66 128 - - ch3 CH2O.COPh 71 168 - - CH, CH2O.CO(2-Br.CgH4) 81 158 - - CH, ch2o.co.cooc2h5 25 170 - - CH, CH2O.CO.CH2Br 41 141 - - CH3 CH, ch2o.co.ch2ci 34 152 - - CH2O.CO(CH)2COOH 85 117 63 121 ch3 CH2O.CO.NH.CH3 46 146 - - ch3 CH2O.CS.NH.CH3 47 146 - - CH, CH,0.C0.NH(CH,),CH, 57 132 - - CH3 CH2O.CO.NH(4-Br.CgH4) 76 119 61 138 CH3 CH20.C0.NHPh 82 121 49 138 ch3 ch2och3 27 164 - - ch3 CH2OCH2Ph 54 140 - - ch3 ch2och2.c=ch 21 145 - - ch3 CH,O(CH,),CH, 64 133 - - ch3 CH,OCH,(2-Br.CfiH4) 89 122 56 153 CH3 CH0 48 140 - - H ch2oh 65 126

Claims (15)

CLAIMS:

1. A derivative of M.139,603 of the formula I wherein:5 M + is an alkali metal, alkaline earth metal, ammonium, alkylammonium or hydroxyalkylammonium cation; R 1 is a hydrogen atom, a 1-6C alkyl radical or a phenyl (1-6C)alkyl radical optionally substituted by halogen, cyano or 1-8C alkyl, alkoxy, halogenoalkyl, 10 halogenoalkoxy or alkanoylamino radicals; and 2. 3 3 R is a radical of the formula -CHjOR wherein R is a 1-6C alkyl, 3-6C alkenyl, 3-6C alkynyl, 2-6C alkoxycarbonyl or 1-6C alkylamino-(2-6C)alkyl radical or a phenyl(1-6C)alkyl radical bearing an optional substituent 15 as defined above; or a radical of the formula -CH 2 O.COR 4 wherein R 4 is an alkyl, (cycloalkyl)alkyl, halogenoalkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, mono- or di-alkylaminoalkyl, alkoxycarbonylalkyl, carboxyalkyl, carbamoylalkyl, N5 alkylcarbamoylalkyl or Ν,Ν-dialkylcarbamoylalkyl radical wherein each alkyl contains up to 6 carbon atoms, and wherein, in each complex radical containing an alkyl . part, the said alkyl part contains 1 to 6 carbon atoms, and wherein each cycloalkyl radical or cycloalkyl part of 10 a (cycloalkyl)alkyl radical contains 3 to 7 carbon atoms, or a phenyl, naphthyl or phenyl(1-6C)alkyl radical each bearing optional substituents as defined above; or a radical of the formula -CHjO.CO.COR 5 wherein R 5 is an amino, mono- or di-(1-6C)alkylamino, or 1-6C alkoxy 15 radical, or a phenyl, naphthyl or phenyl(1-6C)alkyl radical each bearing optional substituents as defined above; or a radical of the formula -CH_O.CX.NR 6 R 7 wherein X is g “ 7 an oxygen or sulphur atom and R and R , which may be the 20 same or different, are each a hydrogen atom, a 1-6C alkyl radical or a phenyl, naphthyl or phenyl(1-6C)alkyl radical each bearing optional substituents as defined above; or 1 2 when R is a hydrogen atom, R is a hydroxymethyl 25 radical.

2. A derivative as claimed in claim 1 wherein any of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 is an alkyl radical of 1 to 3 carbon atoms or is a complex radical containing an alkyl part which contains 1 to 3 carbon atoms or R is an 30 alkenyl or alkynyl radical of 3 carbon atoms.

3. A derivative as claimed in claim 1 wherein M + is a sodium, lithium, potassium, calcium, mangnesium, zinc, ammonium, mono-, di-, tri- or tetraalkylammonium ion wherein each alkyl is of 1 to 10 carbon atoms, or mono-, di- or tri-(hydroxyalkyl)ammonium ion wherein each hydroxyalkyl is of 1 to 10 carbon atoms.

4. A compound as claimed in claim 1 wherein:1 2 R is hydrogen and R is hydroxymethyl.

5. λ compound as claimed in claim 1 wherein: 1 2 R is methyl and R is methoxymethyl; R x is methyl and R z 1 2 R is methyl and R R is methyl and R is propargyloxymethyl; is benzoyloxymethyl; is 2-bromobenzoyloxymethyl; R 1 is methyl and R 2 is chloroacetoxymethyl; R x is methyl and R' R is methyl and R' is ethoxalyloxymethyl; or is N-methylthiooarbamoyloxymethyl.

6. A process for the manufacture of a derivative as 2 claimed in claim 1 wherein R is a radical of the formula 3 -CH.OR as defined in claim 1, which comprises the 2 1 reaction of a compound of the formula I wherein R has 2 any of the meanings defined in claim 1 and R is a hydroxymethyl radical, with a compound of the formula RZ, wherein R has the meaning defined in claim 1 and Z is a leaving group, in the presence of a stong base.

7. A process for the manufacture of a derivative as claimed in claim 1 wherein R 1 is other than hydrogen, and R 2 is a radical of the formula -CH.O.COR 4 , -CH.O.CO.COR 5 fi 7 * * or CHjO.CX.NR R as defined in claim 1, which comprises the reaction of a compound of the formula X wherein R 1 is an alkyl or optionally substituted phenylalkyl radical and R is a hydroxymethyl radical, with an acylating : agent derived from a carboxylic acid of the formula R .COOH or R CO.COOH wherein R and R have the meanings defined in claim 1, with an acid anhydride, with a mixed anhydride, or with a carboxylic acid of the formula R 4 .C00H or R 5 CO.COOH in the presence of a carbodiimide; whereafter:for those compounds wherein R is a radical of the 4 4 formula -CH-O.COR in which R is a hydroxyalkyl radical, a compound of the formula (I) wherein R is a radical of the formula -CHjO.COR 4 in which R 4 is a (t-alkoxy)-alkyl radical obtained as described above is reacted with an acid; or for those compounds wherein R is a radical of the 4 4 formula -CHgO.COR in which R is an alkylaminoalkyl or dialkylaminoalkyl radical, the corresponding aminoalkyl compound obtained as described above, is reacted with an alkyl halide or; for those compounds wherein R is a radical of the 4 4 formula -CH 2 O.COR in which R is an alkoxycarbonylalkyl radical, the corresponding carboxyalkyl compound, obtained as described above, is reacted with a diazoalkane, or a salt of the corresponding carboxyalkyl compound obtained as described above, is reacted with a diazoalkane, or a salt of the corresponding carboxyalkyl compound obtained as described above, is reacted with an alkyl halide; or for those compounds wherein R is a radical of the formula -CH 2 O.COR 4 in which R 4 is a carbamoylalkyl, Nalkylcarbamoylalkyl or Ν,Ν-dialkylcarbamoylalkyl radical, a corresponding carboxyalkyl or alkoxycarbonylalkyl compound obtained as described above, is reacted with ammonia or a mono- or dialkylamino respectively; or for those compounds wherein R_ is a radical of the formula -CHjO.CO.COR in which R is an amino, monoalkylamino or dialkylamino radical, a corresponding compound wherein R 5 is a hydroxy or alkoxy radical, obtained as described above, is reacted with ammonia or a mono- or di-alkylamine respectively; or for those compounds wherein R is a radical of the fi Ύ fi formula -CH_O.CX.NR R wherein R is a hydrogen atom and 7 X and R have the meanings defined in claim 1, a corresponding compound of the formula (X) in which R is a hydroxymethyl radical, obtained as described above, is reacted with an isocyanate or isothiocyanate of the formula R 7 .NCX; or for those compounds wherein R is a radical of 6 7 the formula -CH_O.CX.NR R wherein X has the meaning * 6 7 defined in claim 1 and either one or both of R and R is other than a hydrogen atom, a corresponding compound wherein either or both of R and R is hydrogen atom obtained as described above, is reacted with a halogen 6 7 compound R . halide or R . halide. 8

8. A process for the manufacture of a derivative as 1 2 claimed in claim 1 wherein R is a hydrogen atom and R is a hydroxymethyl radical, which comprises the culturing of Streptomyces lonalsporoflavus NCIB 11426 or a variant or mutant thereof in an aqueous nutrient medium, containing a source of assimilable carbon, under shaken aerobic conditions at a temperature of between 22 and 32°C., extracting the fermentation mixture with a waterimmiseible organic solvent, and isolating from the organic extract the product which has Rj, » 0.43 on thinlayer chromatography on silica plates (Merck's Kieselgel 60F-254- trade mark), 0.25mm. thickness, eluting with a mixture of diethyl ether, methanol and formic acid in the proportion 95:4:1 by volume.

9. A composition which comprises a derivative of 5 M.139,603 as claimed in claim 1 together with a solid or liquid edible non-toxic diluent or carrier.

10. A method for use in the husbandry of domestic ruminant animals for increasing the efficiency of their food conversion, which comprises orally administering to 10 the animals a derivative of H.139.603 as claimed in claim 1.

11. A derivative according to claim 1, substantially as hereinbefore described with particular reference to Examples 1-9 of the accompanying Examples. 15

12. A process for the manufacture of a derivative according to claim 1, substantially as hereinbefore described with particular reference to Examples 1-9 of the accompanying Examples.

13. A derivative according to claim 1, whenever manu20 factured by a process claimed in a preceding claim.

14. A composition according to claim 9, substantially as hereinbefore described with particular reference to Examples 10 and 11 of the accompanying Examples.

15. A method according to claim 10, substantially as 25 hereinbefore described with particular reference to Example 12 of the accompanying Examples.